Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the claims.
Advances in understanding of human diseases at molecular level have led to possibility of treating human diseases by introducing genes into specific cells of patients. Gene therapy offers a great promise for modern medicine (Mulligan 1993) (Anderson 1984). The first step of gene therapy is to transduce specific target cells. There are two basic ways to deliver genes. In ex vivo therapy, cells from certain tissues, such as hematopoietic cells, are removed from patients and are infected with vectors which carry therapeutic genes. Cells expressing the therapeutic genes are then returned into patients. Vectors are directly administrated to patients in in vivo therapy. This approach is especially useful when it is difficult to isolate cells from tissues and to be infected in vitro. Advantages with retroviruses include long-term stability of therapeutic genes because viral genome is inserted into human chromosomes.
To engineer retroviruses for gene therapy, viral genes are deleted from viral genome, and an exogenous gene is inserted instead into viral genome. Plasmids containing a viral genome is introduced into a packaging cell line which provides viral components necessary to make viral particles (Cone and Mulligan 1984; Mann et al. 1983; Miller et al. 1985; Sorge et al. 1984; Watanabe and Temin 1983; Markowitz et al. 1988a; Markowitz et al. 1988b). Virions produced from the packaging cell line are used to transduce target cells. In the first step of viral infection, envelope proteins on retroviruses interact with receptors on target cells, which lead to a series of events resulting in fusion of viral membrane and cellular membrane. Viral core containing viral genomes with exogenous genes thus enters cells. Based on their host range, retrovirus vectors commonly used in human gene therapy are classified as ecotropic or amphotropic. Although ecotropic virus can only infect murine cells, modification of envelope protein, such as inserting a ligand epitope for a specific receptor, will expand or change host range, resulting in infection of human cells expressing that particular receptor (Kasahara et al. 1994). Amphotropic virus can infect both murine and human cells. One problem associated with retrovirus vectors is low transduction efficiency. For example, amphotropic virus has been used to infect hematopoietic cells, and transduction efficiency is low. To circumvent this problem, viruses pseudotyped with envelope proteins from other viruses have been tested. For example, Vesicular stomatitis virus (VSV) G protein and Gibbon ape leukemia virus (GALV) envelope protein have been studied in pseudotyping murine leukemia viruses (Hopkins 1993; Ory et al. 1996; Sharma et al. 1996; Wang et al. 1996) (Lam et al. 1996). In general, however, the titers of those vector stocks are still low, preventing successful clinical application of gene therapy in treating human diseases.
Gene therapy holds great promise in modern medicine. Advances in understanding of genetic bases of human diseases make it possible to treat human diseases by transferring normal genes into specific cells of patients. It has been proposed to use gene therapy to treat any human diseases, genetic and acquired (Anderson 1984; Mulligan 1993). Significant advances have been made to develop protocols to deliver exogenous genes into human cells. Vectors, vehicles used to deliver genes, include retroviruses and other viruses. Retroviral vectors, by far, the most extensively studied among viral vectors offer several advantages over other viral vectors, specially in ex vivo strategy of gene therapy. A retroviral vector can transduce any human cells, and lead to a long-term expression of exogenous gene.
To achieve therapeutic effects with gene therapy technology, vectors which express exogenous genes at a level sufficient to achieve therapeutic effects are required. A great deal of effort has been devoted to identify nucleic acid molecule sequences important for gene expression and to incorporate such nucleic acid molecules in vectors to thereby develop vectors which can express therapeutic genes at high levels (Leboulch et al. 1994; Leboulch et al. 1995; Takekosh et al. 1995). In some instances, regulated expression of the therapeutic genes is required (Cone et al. 1987). Retroviruses, offer an excellent choice to introduce exogenous genes into cells because of their ability to infect any kind of cells. In infected cells, retroviral genomes are inserted into host chromosomes, resulting in a long term expression of exogenous genes.
The second important aspect of gene therapy using a retrovirus as vector is the development of safe packaging cell lines (Cone and Mulligan 1984; Mann et al. 1983; Miller et al. 1985; Sorge et al. 1984; Watanabe and Temin 1983; Markowitz et al. 1988a; Markowitz et al. 1988b). In packaging cells, viral components are made and are able to assemble into viral particles. A vector carrying a therapeutic gene and a retroviral packaging signal is introduced into a packaging cell line and is packaged into viral particles. Retrovirus formed in such a way can be used to transduce target cells. To make safe packaging cell lines, viral proteins, gag and pol are expressed from a plasmid, and the envelope is expressed from another plasmid.
The chance of generating wild type virus through recombination is extremely low (Markowitz et al. 1988a; Markowitz et al. 1988b). Because a viral vector contains only a packaging signal, and does not encode any viral proteins, there is only one round of infection.
To successfully apply retroviral gene therapy in treatment of human diseases, several technical problems have to be solved. One of the difficulties involved is low transduction efficiency of retroviral stocks, which is addressed extensively in this study. Amphotropic retrovirus is widely used in gene therapy because of its ability to infect human cells. In transduction of human lymphocytes using retroviruses with amphotropic envelopes, however, transduction efficiency is relatively low. To improve gene therapy efficiency, different viral envelope proteins have been studied for their application in gene therapy. Gibbon Ape leukemia virus envelope virus envelope, for example, has been used to pseudotype murine leukemia viral vector (Lam et al. 1996). Vesicular stomatitis virus (VSV) G protein is another alternative envelope protein used in gene therapy (Hopkins 1993; Porter et al. 1996; Sharma et al. 1996; Wang et al. 1996). It has been shown that retroviral vectors pseudotyped with G protein can transduce human lymphocytes with much higher efficiency than amphotropic retroviral vectors.
In treating human diseases, sometimes specific targeting of certain cell types is required. Different strategies have been used to modify envelope proteins on vectors. For example, a ligand epitope is inserted into the envelope, which enables virus to infect specific cell type (Kasahara et al. 1994). In general, such modifications result in low infection efficiency.
Polybrene, a chemical compound, is often used to increase viral infectivity of retrovirus. In several cases, instead of boosting transduction efficiency, it actually decreases viral transduction efficiency. For example, plates coated with a fibronectin fragment are often used to isolate human stem cells. Polybrene, however, because of its negative charges, will decrease efficiency of fibronectin. Thus, alternatives are needed for enhancing retroviral infectivity in such circumstances.
Discovery of the Envelope-interacting Proteins (EIPs)
Understanding of the basic mechanism used by retroviruses to enter cells will facilitate application of retroviruses as vectors for gene therapy. Envelope proteins of retroviruses including Mo-MLV contain two subunits: the surface protein (SU) and the transmembrane protein (TM). SU mediates binding of the virus to host cells by interacting with specific viral receptors on a host cell surface, which triggers a complex process leading to fusion of viral and cellular membranes mediated by TM (Hunter and Swanstrom 1990; Marsh and Helenius 1989). These early events of viral infection are poorly understood. Several lines of evidence suggest that host factors are involved in these early events of viral infection. Recently the second receptors for human immunodeficiency virus (HIV) have been identified and cloned (Deng et al. 1996; Dragic et al. 1996; Feng et al. 1996). It has also been suggested that host surface proteins bind to TM proteins of HIV-1 and HIV-2 (Chen et al. 1992; Ebenbichler et al. 1993; Ebenbichler et al. 1996).
Enveloped animal viruses may use similar strategies to enter cells. Their envelope proteins are remarkably similar in structure. For instance, crystal structure of hemaegglutinin of influenza virus is very similar to that of retrovirus envelope (Fass et al. 1996; Fass and Kim 1995). The TM proteins of retroviruses share many structural similarities. For instance, at linear sequence level, there is a stretch of hydrophobic amino acids at the N-terminus that is believed to be involved in the fusion of viral and cellular membranes. Another feature is the leucine zipper motif in the middle of TM proteins (Delwart and Mosialos 1990; Gallaher et al. 1989). They also share structural similarities (Blacklow et al. 1995; Fass et al. 1996; Fass and Kim 1995; Lu et al. 1995).
The functions of TM in viral replication have been studied. It has been shown that TM of Mo-MLV envelope forms oligomers using the yeast two-hybrid system. Deletion and mutational analysis indicate that the putative leucine zipper motif in the extracellular domain of TM is necessary and sufficient for the binding and that the first three repeats of the leucine zipper-like motif are the most important in mediating the interaction (Li et al. 1996).
The present invention provides two cellular proteins have been identified by their ability to interact with transmembrane (TM) protein of Moloney murine leukemia virus (Mo-MLV). They are termed envelope-interacting proteins (EIPs) (Table 1). The studies presented infra show that EIP-1 and EIP-3 can interact with TM protein in the yeast two-hybrid system. In an in vitro binding assay, EIP-1 and EIP-3 can directly bind to ecotropic retrovirus.
To test if the binding of EIPs to virus affects viral infectivity, viruses were incubated with EIPs prior to infection. It was found that EIP-1 and EIP-3 can significantly increase viral transduction efficiency of ecotropic retrovirus using NIH3T3 cell as a target. EIP-1 was also examined to determine if it could increase the infectivity of amphotropic virus, because TM proteins of both ecotropic and amphotropic viruses are basically the same. It was found that EIP-1 significantly enhances infectivity of amphotropic virus using NIH3T3 cells. Similar results were obtained using Hela cells, a human cell line, as target cells.
The present invention provides genes designated EIP-1 and EIP-3 which encode envelope-interacting proteins, EIP-1 and EIP-3, respectively. EIP-1 and EIP-3 proteins interact with the TM envelope to enhance retrovirus infectivity (titer) when these proteins are added to a virus preparation, thereby providing an alternative to increase efficiency of retroviral gene delivery. The proteins provided by the present invention overcome the above-described problems associated with polybrene by increasing stimulation of gene transfer. Accordingly, EIP-1 and EIP-3 of the present invention also provides methods to enhance viral transduction in gene therapy.
This invention provides an isolated nucleic acid molecule encoding a envelope-interacting protein-1. This invention provides an isolated nucleic acid molecule which encodes an EIP-1 comprising an amino acid sequence as set forth in FIGS. 2A-2D.
This invention provides a fusion protein comprising an EIP-1 or a fragment thereof and a second peptide.
This invention provides a vector comprising the isolated nucleic acid molecule, wherein the encoded mammalian EIP-1 comprises the nucleic acid sequence set forth in FIGS. 2A-2D. This invention provides a plasmid designated pCGN-EIP-1 (ATCC Designation No. 209885).
This invention provides a method of producing an EIP-1, which comprises growing a host cell comprising vectors of the invention under suitable conditions permitting production of the EIP-1.
This invention provides an isolated nucleic acid molecule encoding a envelope-interacting protein-3. This invention provides an isolated nucleic acid molecule which encodes an EIP-3 comprising an amino acid sequence as set forth in FIGS. 3A-3E.
This invention provides a fusion protein comprising an EIP-3 or a fragment thereof and a second peptide.
This invention provides a vector comprising the isolated nucleic acid molecule, wherein the encoded mammalian EIP-3 comprises the nucleic acid sequence set forth in FIGS. 3A-3E. This invention provides a plasmid designated pCGN-EIP-3 (ATCC Designation No. 209884).
This invention provides a purified mammalian EIP-1.
This invention provides a protein designated EIP-1 comprising substantially the amino acid sequence set forth in FIGS. 2A-2D.
This invention provides a protein designated EIP-1 having the amino acid sequence set forth in FIGS. 2A-2D.
This invention provides a purified mammalian EIP-3.
This invention provides a protein designated EIP-3 comprising substantially the amino acid sequence set forth in FIGS. 3A-3E.
This invention provides a protein designated EIP-3 having the amino acid sequence set forth in FIGS. 3A-3E.
This invention provides an oligonucleotide comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a unique sequence included within the sequence of the isolated nucleic acid molecule encoding mammalian EIP-1, wherein the nucleic acid molecule comprises the nucleic acid sequence set forth in FIGS. 2A-2D.
This invention provides an oligonucleotide comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a unique sequence included within the sequence of the isolated nucleic acid molecule encoding mammalian EIP-3 wherein the nucleic acid molecule comprises the nucleic acid sequence set forth in FIGS. 3A-3E.
This invention provides a monoclonal antibody directed to an epitope of an EIP-1.
This invention provides an antibody capable of binding to the EIP-3 having the amino acid sequence set forth in FIGS. 3A-3E or to a fusion protein thereof.
This invention provides a monoclonal antibody directed to an epitope of an EIP-3.
This invention provides a method of increasing transduction efficiency of a retrovirus on target cells comprising: a) incubating an envelope-interacting protein with a retrovirus; and b) transducing the target cells with the retrovirus.
This invention provides a method of increasing transduction efficiency of a retrovirus on a target cell comprising: a) incubating an envelope-interacting protein with a target cell; and b) transducing the target cell with a retrovirus.
This invention provides a method of treating a patient with a therapeutic gene comprising: a) incubating a transducing virus with an effective amount of an envelope-interacting protein effective to enhance retroviral infectivity; and b) transducing target cells of the patient with the resulting virus of step (a) bound to the envelope-interacting protein comprising a therapeutic gene, thereby treating the patient with the therapeutic gene.
This invention provides a method of treating a patient with a therapeutic gene comprising: a) incubating a transducing virus with an effective amount of an envelope-interacting protein effective to enhance retroviral infectivity; and b) transducing target cells of the patient with a retroviral virion plus the envelope-interacting protein bound thereto, comprising a therapeutic gene, thereby treating the patient with the therapeutic gene.
This invention provides a method of treating a patient with a therapeutic gene comprising: a) incubating a retroviral virion comprising a therapeutic gene with an effective amount of an envelope-interacting protein to permit enhanced binding of the envelope-interacting protein to the virion; and b) transducing target cells of the patient with the envelope-interacting protein bound virion comprising the therapeutic gene, thereby treating the patient with the therapeutic gene.
This invention provides a pharmaceutical composition comprising an envelope-interacting protein bound retroviral virion comprising a therapeutic gene and a pharmaceutically acceptable carrier capable of passing through a cell membrane. This invention provides a pharmaceutical composition comprising an amount of an envelope-interacting protein bound retroviral virion comprising a therapeutic gene effective to enhance retroviral infectivity of target cells and a pharmaceutically acceptable carrier capable of passing through a cell membrane.
This invention provides a method of treating an abnormality in a subject, wherein the abnormality is alleviated by the administering to the subject an effective amount of any of the above-described pharmaceutical compositions effective to introduce high titers of a therapeutic gene to the subject, thereby treating the abnormality in the subject.